To identify blood proteins with altered abundances in patients with early stage Lyme disease as compared to healthy controls with the intent of identifying potential Lyme diagnostic markers, selected proteins from two categories were monitored by SRM: 1) key innate immunity regulators including acute phase proteins, reflecting the fact that LD is an acute inflammatory disease; and 2) organ-specific blood proteins predominantly expressed in organs and tissues known to be affected by B. burgdorferi, i.e., liver, heart, brain and skin. In all, 174 peptides (representing 124 human proteins) were measured in individual serum samples from 2 independent Lyme disease patient cohorts. Each sample set contains 30-40 confirmed Lyme disease patients (each with 4 time points) and 20 healthy controls (each with 2 time points) from baseline to 4-6 years years post-treatment. 90 proteotypic peptides from 73 targeted proteins were successfully detected and quantified. Six proteins (APOA4, C9, CRP, CST6, PGLYRP2 and S100A9) were confirmed to show significantly altered serum levels in patients at time of presentation. A 9-protein panel was also identified and verified.